AUTONOMOUS UNIVERSITY OF MADRID DEPARTMENT OF MOLECULAR BIOLOGY
Inflammation and Skin Cancer Mediated Through c-Fos/AP-1
EVA MARÍA BRISO DE MONTIANO
MADRID, 2013
AUTONOMOUS UNIVERSITY OF MADRID FACULTY OF SCIENCES
DEPARTMENT OF MOLECULAR BIOLOGY
Inflammation and Skin Cancer Mediated Through c-Fos/AP-1
Doctoral thesis submitted to the Autonomous University of Madrid for the degree of Doctor of Philosophy by
M.Sci. in Molecular Biomedicine, Eva María Briso de Montiano
Thesis Director
Prof. Dr. Erwin F. Wagner
GENES, DEVELOPMENT AND DISEASE GROUP F-BBVA-CANCER CELL BIOLOGY PROGRAMME SPANISH NATIONAL CANCER RESEARCH CENTRE
This thesis, submitted for the degree of Doctor of Philosophy at the Autonomous University of Madrid, has been completed in the Genes, Development and Disease
Laboratory a the Spanish National Cancer Research Centre (CNIO), under the supervision of Prof. Dr. Erwin F. Wagner
This work was supported by the following grants and fellowships:
La Caixa/CNIO International PhD Fellowship. 2008 Call. Eva Briso de Montiano
ERC Advanced Grant: Erwin F. Wagner
Fundación Banco Bilbao Vizcaya (F-‐BBVA) -‐ CNIO Cancer Cell Biology Program
As you set out for Ithaka hope your road is a long one, full of adventure, full of discovery.
Laistrygonians, Cyclops,
angry Poseidon—don’t be afraid of them:
you’ll never find things like that on your way as long as you keep your thoughts raised high, as long as a rare excitement
stirs your spirit and your body.
Laistrygonians, Cyclops,
wild Poseidon—you won’t encounter them unless you bring them along inside your soul, unless your soul sets them up in front of you.
Hope your road is a long one.
May there be many summer mornings when, with what pleasure, what joy,
you enter harbors you’re seeing for the first time;
may you stop at Phoenician trading stations to buy fine things,
mother of pearl and coral, amber and ebony, sensual perfume of every kind—
as many sensual perfumes as you can;
and may you visit many Egyptian cities
to learn and go on learning from their scholars.
Keep Ithaka always in your mind.
Arriving there is what you’re destined for.
But don’t hurry the journey at all.
Better if it lasts for years,
so you’re old by the time you reach the island, wealthy with all you’ve gained on the way, not expecting Ithaka to make you rich.
Ithaka gave you the marvelous journey.
Without her you wouldn't have set out.
She has nothing left to give you now.
And if you find her poor, Ithaka won’t have fooled you.
Wise as you will have become, so full of experience, you’ll have understood by then what these Ithakas mean.
Ithaka. KP Kavafis
Dedicated to my family In memory of my grandmother
A cknowledgements
This doctoral thesis is the result of my research spanning the past four years of my life and would not have been possible without the aid, support and contribution of many people to whom I wish to express my gratitude here.
I want to thank Dr. Erwin Wagner for giving me the great opportunity to carry out my PhD in his group, as well for scientific guidance, discussion of my project and for allowing me to meet internationally recognized scientists and discuss my project with them.
Very special thanks to Dr. Juan Guinea-‐Viniegra, not only for beeing a very good friend of mine but also for his unconditional support, scientific guidance, his patience, for reviewing this thesis and for everything you have taught me during my PhD.
I am very grateful to all the members of the Genes, Development and Disease group and the CCB Program, for creating an inspiring atmosphere in the lab. Especially, I want to thank my neighbors, Francy and Jochen, for those fun moments we spent while pipetting, Marta and María Martín for your help prepping the tails. I want to thank María Jiménez and Ana Guio for organizing the lab.
I want to thank Dr. Mercedes Rincón because of the big support and motivation you have given me. I admire your passion and dedication to science. Above all, because my interest for science started in your lab when I was still an undergraduate student at the University of Vermont.
I would like to acknowledge the members of my PhD committee, Dr. Fiona Watt, Dr. Ángel Nebreda and Dr. Marisol Soengas for scientific guidance and for making possible all those meetings during my PhD.
I want to thank the collaborators, Dr. Peter Angel, for carrying out the microarray analyses and for scientific discussion. Also, Dr. Peter Petzelbauer, for sharing his knowledge as a dermatophatologist with us, for travelling to Madrid to discuss our projects and for providing me with so many precious human samples.
I want to thank the 2008 La Caixa fellows. Matt, Jarek S., Aga, Kerstin, Marta, Sara, Miljana, Jarek C., and especially to Sara Mainardi, for being such a good friend inside and outside CNIO, for all those moments spent together outside CNIO, enjoying the life as it
is. I also want to thank Miljana, for being a great roomie and I want to thank Marta Nasila for beeing my first friend in the lab.
I would like to thank many people from CNIO. Thank you Lina, for all those breakfasts and moments we spent together during this time. I want to thank Ultan for helping me with the Flow Cytometer and for all the music you have given to me and for the Primavera Sound 2012! I want to thank Carlitos, for so many concerts we went together. I want to thank my "library friends" Laura and Bárbara, for being next to me during this time while I was writing the thesis and for you immense support.
Quiero agradecer todo el apoyo que me dan mis amigos, Cris Alonso., Elena, Marta, Bego, Jesús, Cristina Bosch. Gracias por todos esos momentos de cenas y sesiones de cine. Tampoco quiero olvidarme de mis amigas las biólogas, repartidas por todo el mundo. Asun, Bea Buitrago, Irene Ureña, Laura, Martita, Ana, Bea Cabanillas. Seguid así.
No dejéis de luchar por aquello en lo que creéis. Gracias Mª Luz por enseñarme la luz al final del tunel.
Lo más especial siempre llega al final. No quiero olvidarme de mi familia. De mis tios, tias y primos, por ese grupo tan maravilloso de primos que siempre debe seguir unido (¡Viva Mamblas!). No me quiero olvidar de Tere, porque para mí has sido una persona muy importante en mi vida. Quiero agradecerle a mi abuela Satur todo el amor que me dio durante su vida, porque nunca te olvidaré por muy lejos que te hayas ido. y porque me hubiera encantando que estuvieras aquí el día de la defensa de mi tesis doctoral.
Quiero agradecer inmensamente a mis hermanas, Marta e Irene, porque sois las mejores hermanas del mundo, las mejores amigas y porque me encanta compartir tantos momentos juntas ya sea en la vida cotidiana o de viaje. Porque me encanta hablar con vosotras.
Por último no tengo palabras para escribir lo agradecida que estoy a mis padres, Manuel y Feli. Gracias por vuestro apoyo incondicional durante estos cuatro años, por vuestro cariño y por vuestro inmenso corazón.
T able of C ontents
TABLE OF CONTENTS
SUMMARY...5
RESUMEN...9
ABBREVIATIONS ... 13
INTRODUCTION... 17
1. THE SKIN ...19
1.1. The Dermis ...19
1.2. The Epidermis ...19
Figure I1.... 20
1.3. Epidermal and dermal crosstalk ...21
1.4. Epidermal Stem Cells ...22
1.5. Human skin versus mouse skin...23
2. SKIN CANCER ...23
2.1. Basal Cell Carcinoma (BCC)...24
2.2. Cutaneous Squamous Cell Carcinoma (SCC) ...24
Figure I2.... 25
3. THE ACTIVATOR PROTEIN 1 (AP-‐1) TRANSCRIPTION FACTOR ...26
3.1. Structure and function...26
Figure I3.... 26
3.2. AP-1 signal transduction...27
Figure I4.... 27
3.3. Biological functions of AP-1 in mice...28
3.3.1. Biological functions of AP-‐1 in skin... 28
3.3.2. AP-‐1 functions in tumorigenesis ... 28
3.3.2.1. AP-‐1 functions in skin tumorigenesis... 30
4. TUMOR MICROENVIRONMENT...31
4.1. Extracellular Matrix (ECM) ...31
4.2. Matrix metalloproteases (MMPs)...32
4.3. Cancer Associated Fibroblasts (CAFs)...33
4.4. Inflammation and Cancer...34
4.4.1. Tumor-‐protective inflammation... 34
4.4.2. Tumor-‐promoting inflammation... 35
4.4.3. Skin inflammation and cancer ... 36
Figure I5.... 36
Table I1.... 38
OBJECTIVES ... 41
OBJETIVOS ... 45
MATERIALS AND METHODS... 49
1. MICE ...51
1.1. Study approval...51
1.2. Generation of the tet-switchable c-fos allele ...51
1.3. Mouse lines ...51
1.3.1. c-‐FosEp-‐tetON and c-‐FosEp-‐tetOFF mouse lines... 51
1.3.2. c-‐FosEp-‐tetON; Rag1-‐/-‐ mouse line... 52
1.4. Mouse Genotyping...52
Table M1.... 52
1.5. Mouse treatments...53
1.5.1. Doxycycline treatment... 53
1.5.2. Chemical carcinogenesis ... 53
1.5.3. Anti-‐inflammatory treatment ... 53
1.5.4. MMP inhibitory treatment ... 53
1.6. Skin barrier assays...53
1.6.1.Transepidermal water loss (TEWL) in vivo epidermal barrier assay... 53
1.6.2 Toluidine Blue in vivo epidermal barrier assay... 54
2. HISTOLOGICAL ANALYSIS ...54
2.1. Fresh frozen tissue ...54
2.2. Formalin-fixed paraffin-embedded tissue ...54
2.3. Human samples and Tissue Microarray (TMA) preparation...55
2.4. Nile Red staining...55
3. PROTEIN ANALYSES...55
3.1. Protein extraction and quantification ...55
3.2. Immunoblotting...56
3.3. Chromatin Immunoprecipitation (ChIP) ...56
4. RNA ANALYSES...56
4.1. RNA extraction from tissues or cells ...56
4.2. RNA extraction from FACS-sorted cells ...57
Table M2.... 57
Table M3.... 58
4.3. Genome-wide expression analyses...58
5. FLOW CYTOMETRY...59
5.1. Back skin-specific protocol...59
5.2. Lymph node-specific protocol...60
5.3. Flow cytometry analyses...60
5.4. FACS sorting...61
6. CELL CULTURE ...61
6.1. Keratinocyte primary cultures ...61
6.1.1.Primary keratinocytes... 61
6.1.3. E Low Calcium medium ... 62
6.1.4. SCC cell lines... 62
6.1.5. Feeders ... 62
6.4. In vitro proliferation assays...62
6.4.1. Cell Counts... 62
6.4.2. Colony formation assays ... 62
6.4.3. EdU incoroporation assay ... 63
Statistical analyses...63
RESULTS ... 65
1. INDUCIBLE EPIDERMAL C-FOS EXPRESSION IN ADULT MICE LEADS TO EPIDERMAL HYPERPLASIA WITH INCREASED PROLIFERATION ...67
1.1. Inducible keratinocyte-specific c-fos expression: c-FosEp-tetON mouse model...67
Figure 1... 67
Figure 2... 69
1.1.2. c-Fos promotes proliferation in vivo in a non-cell autonomous manner...70
Figure 3... 70
Figure 4... 71
1.1.3. Impaired differentiation upon c-fos expression in vitro ...72
Figure 5... 73
1.2. Inducible keratinocyte-specific c-fos expression: c-FosEp-tetOFF mouse model...74
Figure 6... 74
Figure 7... 75
1.2.1. The epidermal barrier is not affected upon c-fos expression ...76
Figure 8... 77
2. MMP10 AND S100A7A15 ARE TWO NOVEL TRANSCRIPTIONAL TARGET GENES OF C-‐FOS....78
2.1. Genome-wide expression analyses revealed novel target genes of c-Fos in keratinocytes .78 2.2.Validation of c-Fos target genes in vitro...78
Figure 9... 79
2.3. Validation of c-Fos target genes in vivo ...79
Figure 10.... 80
2.4. Mmp10 and s100a7a15 promoter analyses ...81
Figure 11.... 81
3. C-FOS EXPRESSION INDUCES SKIN INFLAMMATION CHARACTERIZED BY CHRONIC CD4 T CELL RECRUITMENT ...82
Figure 12.... 83
Figure 13.... 84
Figure 14.... 85
4. INTERFERING WITH CD4 T CELL RECRUITMENT SIGNIFICANTLY IMPAIRS C-‐FOS-‐ MEDIATED EPIDERMAL HYPERPLASIA...86
Figure 15.... 87
5. BROAD MMP INHIBITION PREVENTS THE DEVELOPMENT OF PRENEOPLASTIC LESIONS UPON C-‐FOS EXPRESSION IN C-‐FOSEP-TETON MICE ...88
Experimental set-‐up. Control and c-‐FosEp-‐tetON mice were treated with Dox and with vehicle or 10mg/kg of TAPI-‐1 injected IP three times a week for 4 weeks... 88
Figure 17.... 89
6. C-‐FOS-‐DEPENDENT SKIN PHENOTYPE IS LARGELY REVERSIBLE ...90
Figure 18.... 91
Figure 19.... 92
7. DMBA-‐INDUCED PAPILLOMA AND SCC DEVELOPMENT IS ACCELERATED BY C-‐FOS...93
7.1. c-FosEp-tetON mice develop invasive SCCs upon DMBA...93
Figure 20.... 94
Figure 21.... 95
Figure 22.... 96
7.2. c-FosEp-tetOFF mice develop highly invasive SCCs upon DMBA ...97
Figure 23.... 97
Figure 24.... 98
7.3. Impaired mmp10 and s100a7a15 expression in c-fos deficient K5-SOS+ tumor-prone mice ...98
Figure 25.... 99
7.4. Sulindac treatment reduces SCC size and number...99
Figure 26.... 100
8. HUMAN SCCS EXPRESS HIGH C-‐FOS PROTEIN LEVELS CORRELATING WITH HIGH MMP10 BUT NOT WITH S100A7 EXPRESSION LEVELS ... 101
Figure 27.... 102
8.1. SCCs but not BCCs express c-Fos and this correlates with MMP10 expression...102
Figure 28.... 103
Figure 29.... 104
8.2. c-FOS protein-expressing SCCs present CD4 T lymphocyte infiltrates...104
Figure 30.... 105
DISCUSSION ...107
1. C-‐FOS IN EPIDERMAL HOMEOSTASIS... 109
2. C-‐FOS TRIGGERS INFLAMMATORY PROCESSES IN THE SKIN THAT LEAD TO THE DEVELOPMENT OF PRENEOPLASTIC LESIONS... 111
3. C-‐FOS TRANSCRIPTIONALLY CONTROLS MMP10 AND S100A7A15 EXPRESSION ... 115
4. C-‐FOS FUNCTIONS IN SKIN CANCER DEVELOPMENT AND PROGRESSION ... 117
Figure 31.... 121
CONCLUSIONS ...123
CONCLUSIONES...129
REFERENCES...135
APPENDIX...151
Table A... 153
S ummary
In this study I describe a novel mechanism by which increased levels of c-‐
Fos/AP-‐1 transcription factor in the epidermis promotes the development of epidermal preneoplastic lesions and eventually, upon 7,12-‐dimethyl-‐benz[a]anthracene (DMBA) treatment, it contributes to the development of skin Squamous Cell Carcinomas (SCCs).
To unravel the function of epidermal c-‐Fos, we have generated a mouse model (c-‐FosEp-
tetON) in which we can inducibly activate the expression of c-fos in the basal layer of the epidermis as well as in other stratified epithelia. I show that inducible c-fos expression triggers innate and adaptive immune responses in the epidermis, particularly, transient recruitment of Gr1+ cells and chronic recruitment of CD4 T lymphocytes. In addition, broad genome-‐wide expression analyses identified two direct and novel transcriptional target genes of c-‐Fos, mmp10 and s100a7a15. Both target genes are involved in inflammatory processes, being able to recruit Gr1+ cells and CD4 T lymphocytes, as well as in the development of cutaneous cancers. Importantly, using a broad matrix metalloprotease (MMP) inhibitor to reduce MMP10 activity in the epidermis, we observed amelioration of the development of the preneoplastic lesions of the skin.
Furthermore, in the absence of mature B and T cells in a Rag1-‐deficient background, a drastic improvement of the disease in FosEp-tetON mice was observed. Moreover, upon DMBA-‐induced H-‐Ras mutations, c-‐Fos is sufficient to promote the development of skin SCCs. Interstingly, in this setting, tumor size, number and burden were significantly reduced upon blockade of inflammatory responses by means of a cyclooxygenase 1 and 2 (COX-‐1/COX-‐2) inhibitory treatment following DMBA treatment and inducible expression of c-fos. Finally, I have seen a strong correlation between human c-‐FOS and MMP10 expression in human SCCs, where they are abundantly expressed, compared to BCCs, where no expression of these two proteins is observed. In addition, a strong correlation between c-‐FOS expression and CD4 T cell infiltrates were observed in human SCCs. This thesis has identified two novel and direct transcriptional targets of c-‐Fos, MMP10 and S100a7a15, clearly involved in mediating CD4 T cell-‐mediated immune responses in the epidermis and thereby contributing to the development of preneoplastic lesions, which upon oncogenic insults, eventually develop into SCCs. Here I propose two new candidate proteins that could be of therapeutic interest to treating cutaneous SCCs.
R esumen
Los resultados de mi proyecto de tesis han dado lugar a la descripción de un nuevo mecanismo mediante el cual el factor de transcripción c-‐Fos/AP-‐1 promueve el desarrollo de lesiones preneoplásicas en la epidermis y en combinación con la aplicación de DMBA, promueve el desarrollo de carcinomas epidermoides (SCCs). Para estudiar la función de c-‐Fos en la epidermis, hemos generado un modelo genético de ratón (c-‐FosEp-
tetON) en el cual se puede inducir la expresión de c-fos en la capa basal de la epidermis y en otros epitelios estratificados. Aquí demuestro que la expresión inducible de c-fos promueve respuestas inmunes innatas y también adaptativas en la piel, fomentando la infiltración de células Gr1+ transitoriamente y de linfocitos T CD4+ de forma crónica.
Mediante análisis de expresión genómica se identificaron dos nuevos genes diana, mmp10 y s100a7a15, ambos implicados en procesos inflamatorios así como también en cánceres de piel. La inhibición de metalloproteasas (MMPs) mediante el uso de un inhibidor, redujo significativamente la progresión de las lesiones preneoplásicas tras la inducción de la expresión de c-fos en ratones c-‐FosEp-tetON. Además, una mejora drástica se observó en el 80% de los casos al deplecionar las poblaciones de linfocitos T y B maduros, mediante el uso de un ratón knock-‐out para Rag1. Asimismo, en esta tesis he demostrado que el factor de transcripción c-‐Fos/AP-‐1 es suficiente para promover el desarrollo de carcinomas epidermoides en ratones tratados con el carcinógeno DMBA.
En este mismo sistema, el bloqueo de procesos inflamatorios en la piel, mediante la inhibición de las enzimas COX1 y COX2, en ratones inducidos con DMBA y c-‐Fos se observa una disminución en el desarrollo de carcinomas epidermoides y aquellos desarrollados son más pequeños. Finalmente, los análisis de muestras humanas de carcinomas epidermoides y carcinomas basales de piel han descubierto la posible implicación de c-‐FOS sólo en los carcinomas epidermoides dado que el 80% de estos tumores presentaba altos niveles de c-‐FOS, mientras que los carcinomas basales no lo expresaban. Finalmente, he observado una correlación entre la expresion de c-‐FOS y de MMP10 en carcinomas epidermoides, MMP10 podría perfectamente ser un gen diana de c-‐FOS en humano como observamos en ratón. Asimismo, se observó correlación entre los niveles de expresión de c-‐FOS en los carcinomas epidermoides y los altos niveles de infiltración de células T CD4+. Esta tesis ha servido para identificar dos genes transcripcionalmente activados por c-‐Fos, mmp10 y s100a7a15, claramente implicados en el desarrollo de procesos inflamatorios mediados por células T CD4+ en la piel y que
promueven inicialmente el desarrollo de lesiones preneoplásicas y eventualmente el desarrollo de carcinomas epidermoides. El desarrollo de fármacos específicos para estas dos dianas puede ser de uso terapéutico en carcinomas epidermoides.
A bbreviations
ABBREVIATIONS
ADAM A disintegrin and metalloproteinase
ADAMTS A disintegrin and metallopreinase with thrombospoindin motifs AP-‐1 Activator Protein 1
ATF Activating transcription factor BCC Basal Cell Carcinoma
BSA Bovine Serum Albumine ChIP Chromatin IP
CTLS Cytotoxic T lymphocytes DC Dendritic cell
DMBA 7,12-‐dimethyl-‐benz[a]anthracene
Dox Doxycycline
ECM Extracellular matrix EdU 5-‐ethynyl-‐2`-‐deoxyuridine EGF Epidermal growth factor
EGFR Epidermal growth factor receptor ERK Extracellular signal regulated kinase FBS Fetal bovine serum
GM-‐CSF Granulocyte monocyte colony stimulating factor HEK Human epidermal keratinocytes
HF Hair follicle
HPV Human papillomavirus
IBD Inflammatory bowel disease IEL Intraepithelial lymphocyte IFE Interfollicular epidermis
IHC Immunohistochemistry
IP Intraperitoneal
JNK Jun N-‐terminal kinase
K1 Keratin 1
K10 Keratin 10
K5 Keratin5
K6 Keratin 6
KGF Keratinocyte growth factor LOH Loss of heterozigosity LOX Lysil oxidase
MAPK Mitogen-‐activated protein kinase
MMP Matrix Metalloprotease
NK Natural Killer
NMSC Non melanoma skin cancer OCT Optimal cutting temperature PBS Phosphate buffered saline
PDAC Pancreatic ductal adenocarcinoma
PTCH1 Patched1
RT Room temperature
RtTA Reverse transactivator
SC Stem cell
SCC Squamous Cell Carcinoma SDS Sodium dodecyl sulfate
SHH Sonic Hedgehog
SLE Systemic Lupus Erythematosus
SMO Smoothened
SOS Son of Seveless
TACE TNFa-‐Converting Enzyme
Tet Tetracycline
TEWL Transepidermal Water Loss Tgf-‐β Transforming Growth Factor Beta TNFα Tumor necrosis factor alpha
TPA 12-‐O-‐Tetradecanoylphorbol-‐13-‐acetate
tTA Transactivator
VEGF Vascular endothelial growth factor
I ntroduction
1. THE SKIN
The skin is the body´s largest organ. It serves as a protective barrier that protects against loss of fluids, physical trauma and invasion by harmful microbes. The skin is divided into two different compartments, the dermis and the epidermis. Interactions between epithelial and mesenchymal cells play a crucial role in the regulation of tissue morphogenesis, homeostasis and repair (Beck and Blanpain, 2012).
1.1. The Dermis
The dermis is the fibrous connective tissue between the epidermis and the subcutaneous fat and is responsible for providing nutrients and physical support to the epidermis (Burr and Penzer, 2005). The most abundant cell type in the dermis is the fibroblast (McLafferty et al., 2012). Fibroblasts synthesize the extracellular matrix and collagen required for normal homeostasis. Type I collagen is by far the most abundant protein in human skin, comprising greater than 90% of its dry weight and the unique physical properties of collagen fibers is to confer structural integrity to skin (Fisher et al., 2008). The dermis also contains blood and lymph vessels, nerve endings, hair follicles and glands. Immune cells also populate the dermis. Some immune cells like mastocytes, histiocytes and γ/δ T lymphocytes are resident in the skin, but other leukocytes like T cells migrate to the skin upon injury or infection (Gebhardt et al., 2011).
1.2. The Epidermis
The epidermis is a stratified epithelium that contains a single inner (basal) layer of proliferative keratinocytes that adhere to the basement membrane, which is rich in extracellular matrix and growth factors, and separates the epidermis from the underlying dermis. Cells in the basal layer are responsible for generating the layers of non-‐dividing cells that undergo a program of terminal differentiation as they move outward and are continually shed from the skin surface (Figure 1) (Fuchs and Nowak, 2008). The balance between proliferation and differentiation is tightly regulated, since the disruption of this balance causes several pathological conditions including inflammation and tumorigenesis (Blanpain and Fuchs, 2009). Indeed, a disruption of the differentiation-‐promoting Notch signaling pathway or a hyperactivation of the EGFR-‐
Ras-‐MAPK signaling pathway in keratinocytes, leads to the development of epithelial tumors (Demehri et al., 2009; Brown et al., 1998).
Figure I1.
The skin and its appendages. Cross-section through mammalian skin and a hair follicle (Fuchs and Raghavan, 2002)
As epidermal keratinocytes exit the basal layer and cease to proliferate, they progress upward through three distinct differentiation stages: spinous layer, granular layer and stratum corneum. The major structural change at the basal-‐to-‐spinous-‐layer transition is the switch from keratin 5 and keratin 14 intermediate filaments in the basal layer to Keratin 1 and Keratin 10 suprabasally. Additional changes occur in the basal/spinous transition, such as downregulation of p63, a member of the p53 family of transcription factors (Dotto, 2009). p63 is expressed in basal cells of all stratified epithelia and is thought to represent a master regulator of the stratification process (Blanpain et al., 2007). Indeed, p63-‐deficient mice die postnatally from severe developmental anomalies, including a lack of stratified epithelia (Mills et al., 1999).
As cells enter the granular layer, the primary cornified envelope protein loricrin is expressed and lamellar granules packed full of lipids appear (Blanpain and Fuchs, 2009). Profilaggrin is also expressed at this time, and soon afterwards, it is proteolytically processed to generate filaggrin, a protein that bundles keratin filaments
into indestructible cables (Aho et al., 2012). As granular cells transit to the stratum corneum, the metabolical activity ceases, and an influx of calcium results in activation of transglutaminases, that initiate glutamyl-‐e-‐lysine crosslinks to produce the cornified envelope, characteristic of this layer (Eckert et al., 2005). The cornified envelope surrounds cells in the stratum corneum and contributes to the skin's barrier function (Simpson et al., 2011). Eventually, keratinocytes in the stratum corneum undergo apoptosis and are released from the surface of the epidermis (Blanpain and Fuchs, 2009).
The epidermis also has the remarkable ability to elaborate the body surface with appendages, which range from hair follicles, nails, oil and sweat glands in mammals to scales and feather in lower vertebrates (Fuchs and Nowak, 2008).
Besides keratinocytes, three other cell types are found in the epidermis:
melanocytes, Langerhans cells and Merkel cells. They are not abundant, but have important functions. Melanocytes are located in the lower part of the epidermis and they synthesize melanin, the pigment that gives skin the natural color (Haass and Herlyn, 2005). Langerhans cells are dendritic cells (antigen-‐presenting immune cells) of the epidermis and they are present in all layers of the epidermis, but are most prominent in the stratum spinosum where they take up and process microbial antigens to become fully functional antigen presenting cells (Romani et al., 2012). Merkel cells are oval receptor cells that have synaptic contacts with somatosensory neurons and these cells are involved in the sensation of light touch (Boulais and Misery, 2007).
1.3. Epidermal and dermal crosstalk
Interactions between mesenchymal and epithelial cells are responsible for complex events such as tissue development, homeostasis and repair (Werner and Smola, 2001). This mutual crosstalk mainly involves growth factors and cytokines. Pioneering studies of Rheinwald and Green demonstrated that normal human epidermal keratinocytes depend on the presence of fibroblasts for efficient growth in tissue culture (Rheinwald and Green, 1975). Later on Rubin, et al identified a fibroblast-‐derived growth factor (FGF), termed Keratinocyte Growth Factor (KGF), that strongly stimulated the proliferation of keratinocytes (Rubin et al., 1989). Moreover, the group of Peter Angel described a paracrine loop in which by using immortalized fibroblasts deficient
for c-‐Jun and JunB, in combination with human primary keratinocytes in the three-‐
dimensional organotypic co-‐culture system, observed that the lack of either one of these transcription factors in fibroblasts severely affected proliferation and differentiation of the overlying normal human keratinocytes (Szabowski et al., 2000). This was due to a direct transcriptional regulation of KGF and GM-‐CSF by Jun/AP-‐1 proteins in fibroblasts.
Furthermore, this crosstalk could also have an impact in epithelial tumorigenesis.
Indeed, recent studies have shown that a disruption in the Notch signaling pathway in the mesenchymal compartment leads to the secretion of soluble factors in an AP-‐1 dependent manner (FGF7, FGF10, CSF1, MMP3 and MMP13). These factors create an appropriate microenvironment in the dermis, also termed as "field cancerization", that promotes epithelial tumorigenesis (Hu et al., 2012).
1.4. Epidermal Stem Cells
The adult epidermis and its appendages undergo continuous renewal and maintain reservoirs of multipotent stem cells (SC). Different stem cell pools have been found in the hair follicle (HF) as well as in the interfollicular epidermis (IFE).
HF stem cells reside in a specialized microenvironment called the bulge. These cells cycle slowly, as revealed by their ability to retain a pulse of nucleotide label following weeks of chase (Alonso and Fuchs, 2003). The bulge is composed of a heterogeneous population of self-‐renewing multipotent cells. Stem cell subpopulations in the bulge exhibit different locations (basal versus suprabasal) and different characteristics, like slowly cycling (quiescent) versus rapidly cycling (Fuchs, 2009). In contrast to the HF, much less is known about stem cells in the IFE. Although lineage-‐
tracing experiments have clearly demonstrated that homeostasis in mouse epidermis is fueled by an independent stem cell population, their origin and localization is still a matter of debate (Beck and Blanpain, 2012).
Stem cells in the basal layer of the epidermis can undergo symmetric and asymmetric cell division. The maintenance of a constant pool of stem cells can be accomplished by one of two distinct types of cell divisions during tissue homeostasis: in asymmetric division, where one daughter remains a SC throughout self-‐renewal, and the other daughter becomes committed to enter a program of terminal differentiation. By
which for SCs would result in the generation of two SCs (self-‐renewal) or two differentiated cells (symmetric differentiation).
1.5. Human skin versus mouse skin
The stratification of mouse skin and human skin is similar—although distinct differences do exist, such as the increased thickness of human skin in comparison with that of mice (Lowes et al., 2007). Mouse skin is heavily populated by hair follicles, whereas the human epidermis is mainly interfollicular; the differences are less striking in skin from the ear and the tail of mice compared with the hairy back skin. Moreover, mouse skin lacks sweat glands and melanocytes in the interfollicular epidermis, but in contrast shows a synchronized hair cycle, rapid epidermal turnover and the presence of intra epidermal γδ T cells (Berking et al., 2002; Jameson et al., 2004; Khavari, 2006;
Wagner et al., 2010). The murine immune system contains particular subtypes of cells, including CD8+ dendritic cells (DCs), dendritic epidermal T cells and natural killer (NK) 1.1+ T cells that are absent in human skin. Despite the obvious differences between mouse skin and human skin, mouse models have been successfully employed to mimic human skin disease in contact hypersensitivity, wound healing, inflammation as well as skin cancer to model monogenic hereditary skin diseases. Clearly, mouse and human skin have many aspects and molecular pathways in common.
2. SKIN CANCER
Skin cancer is the third most common human malignancy and its occurrence has been increasing rapidly over the past decades. An estimated number of 2-‐3 million non-‐
melanoma skin cancer patients and 132,000 patients of melanoma are counted every year (World Health Organization).
Melanoma is the type of skin cancer that arises from the melanocytes, melanin-‐
producing cells located in the basal layer of the epidermis. It is the most dangerous type of skin cancer as it is the leading cause of death from skin disease (Tsao et al., 2012).
10% of melanoma patients have a family history that confers approximately a twofold increase in probability to develop melanoma. Several genes have been identified to predispose to melanoma. Amongst others, mutations in CDKN2A (encoding p16), CDK4,
RB1 lead to the development of melanoma. Other genes such as PTEN or B-‐RAF have been described to promote melanoma (Maubec et al., 2012; Rezze et al., 2012).
Non-‐Melanoma Skin cancer comprises two major types of Skin Cancers, Basal and Squamous Cell Carcinoma (BCC and SCC). Both tumors arise from keratinocytes, but are very different in morphology and in the underlying mechanisms (Colmont et al., 2012).
2.1. Basal Cell Carcinoma (BCC)
It is the most common and least dangerous form of skin cancer (Kasper et al., 2012). It grows slowly, usually on the head, neck and upper torso. BCCs appear on skin exposed to UV light radiation and typically occur in the fourth decade of life and beyond (Kasper et al., 2012). Pathologically, it resembles the keratinocytes in the basal layer of the epidermis (Crowson, 2006). The vast majority of BCCs occur sporadically, but patients with the rare heritable disorder “Basal cell nevus syndrome” have a marked susceptibility to developing BCCs. Several genes of the Sonic Hedgehog (SHH) signaling pathway are frequently mutated (Epstein, 2008). Approximately 90% of sporadic BCCs have identifiable mutations in at least one allele of PTCH1, and an additional 10% have activating mutations in the downstream effector, smoothened (SMO) protein, which renders SMO resistant to inhibition by PTCH1 (Epstein, 2008). Several compounds targeting members of the SHH signaling pathway are being used in clinical trials (Kasper et al., 2012). Removal of tumors using surgery is widely established in less invasive BCCs.
2.2. Cutaneous Squamous Cell Carcinoma (SCC)
Cutaneous Squamous cell carcinoma is the second most common type of human cancer with over 250,000 new cases annually in the USA and it is the second in incidence after BCC. It arises from keratinocytes of the epidermis and oral mucosa. Pathologically, keratinocytes in this type of cancer share features with the squamous cells seen in the outermost layers of the epidermis. Unlike BCCs, cutaneous SCCs are associated with a substantial risk of metastasis (Ratushny et al., 2012). SCC is most commonly found in sun-‐exposed areas. Besides ultraviolet light, other risk factors have been associated with skin SCC, such as arsenic exposure, tobacco and human papilloma virus infection (Brantsch et al., 2008). SCC typically manifests as a spectrum of progressively advanced
malignancies, ranging from a precursor lesion like actinic keratosis to SCC "in situ", invasive SCC and finally metastatic SCC (Figure 4) (Ratushny et al., 2012).
Figure I2.
Histological features of the different stages of human SCC development, from healthy skin to metastatic SCC.
Several syndromes like Xeroderma Pigmentosum or Epidermolysis bullosa have been associated with increased risk to develop SCCs. As with other cancers, SCCs exhibit impaired genomic maintenance that facilitates acquisition of new mutations. p53 is commonly mutated in dysplastic lesions. 40% of SCC in situ harbors p53 mutations, indicating that p53 loss occurrs prior to tumor invasion (Campbell et al., 1993).
Aberrant activation of EGFR (Epidermal Growth Factor) and Fyn, a Src-‐family tyrosine kinase, are seen in human SCCs. Furthermore, amplification and activating mutations of the Ras oncogene have been found in SCCs (Pierceall et al., 1991). 21% of SCCs harbor activating Ras mutations. Of the three Ras genes, Harvey rat sarcoma virus oncogene (H-
Ras) is preferentially mutated in SCCs.
Skin SCC has been extensively modeled by either making use of genetically modified mice, such as K14-‐HPV or the K5-‐SOS mouse models (Arbeit et al., 1994; Sibilia et al., 2000), or by using the two-‐step chemical carcinogenesis protocol (Kemp, 2005). In this protocol, mutations in H-Ras are induced by a single topical dose of a carcinogen, most commonly 7,12-‐dimethyl-‐benz[a]anthracene (DMBA), applied on the back skin.
Repeated topical applications of a tumor promoter, such as TPA give rise to benign neoplastic lesions, which causes sustained hyperplasia (papillomas) and inflammation.
A small percentage of these papillomas progress to malignant invasive squamous cell carcinomas (SCC). In carcinomas, loss of heterozygosity (LOH) and mutations of the tumor suppressor p53 are frequent. More aggressive carcinomas show additional LOH and mutations of the tumor suppressors p19/Arf and p16Ink4a (Kemp, 2005). The use of